INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS-1 PROTEASE BY A C2-SYMMETRICAL PHOSPHINATE - SYNTHESIS AND CRYSTALLOGRAPHIC ANALYSIS

The human immunodeficiency virus type 1 (HIV-1) protease is a potentia l target of acquired immune deficiency syndrome (AIDS) therapy. A high ly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV- 1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has be en determined at 2.3-angstrom resolution by X-ray diffraction techniqu es and refined to a crystallographic discrepancy factor, R (= SIGMA pa rallel-to F(o)\-\F(c) parallel-to/SIGMA\F(o)\), of 0.178. The inhibito r is held in the enzyme active site by a set of hydrophobic and hydrop hilic interactions, including an interaction between Arg8 and the cent er of the terminal benzene rings of the inhibitor. The phosphinate est ablishes a novel interaction with the two catalytic aspartates; each o xygen of the central phosphinic acid moiety interacts with a single ox ygen of one aspartic acid, establishing a very short (2.2-2.4 angstrom ) oxygen-oxygen contact. As with the structures of penicillopepsin bou nd to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka , N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochem istry 31, 5201-14], we interpret this short distance and the stereoche mical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proto n located midway between the two atoms. Under identical assay conditio ns, SB204144 binds approximately 2 orders of magnitude more tightly th an the monohydroxy analog A74704 [Erickson, J., Neidhart, D. J., VanDr ie, J., Kempf, D. J., Wang, X. C., Norbeck, D. W., Plattner, J. J., Ri ttenhouse, J. W., Turon, M., Wideburg, N., Kohlbrenner, W. E., Simmer, R., Helfrich, R., Paul, D., & Knigge, M. (1990) Science 249, 527-33], apparently as a consequence of the stronger hydrogen bonds between th e phosphinate oxygens and the catalytic aspartates. Implications for t he catalytic mechanism of the novel mode of binding of the phosphinate group are discussed.